Maternal Zygotic Transition Research Articles

215 INVESTIGATION OF A PREFERENTIALLY UPREGULATED GENE CLUSTER IN DAY 7 BOVINE EMBRYOS DERIVED FROM RNA SEQUENCING DATA

Successful establishment and maintenance of pregnancy requires optimum conceptus-maternal cross talk. Despite significant progress in our understanding of the temporal changes in the transcriptome of the uterine endometrium, we have only a rudimentary knowledge of the genes and pathways governing growth and development of the bovine conceptus. A recent RNA sequencing study from our group (Mamo et al. 2011 Biol. Reprod. 85, 1143–1151) described the global transcriptome profile of the bovine conceptus at 5 key stages of its pre- and peri-implantation growth (Days 7, 10, 13, 16, and 19) using RNA sequencing techniques. One cluster of genes (n = 1680 transcripts) was preferentially upregulated at Day 7 and subsequently downregulated, suggesting that these genes might be markers of blastocyst formation. The objective of this study was to characterise the pattern of expression of these genes before Day 7 (i.e. from the zygote to blastocyst stage). The list of genes was submitted to DAVID (Database for Annotation, Visualisation, and Integrated Discovery) to take advantage of available ontology information contained therein. The expression of 9 genes belonging to ontologies specifically related to embryo developmental (GINS1, TAF8, ESRRB, NCAPG2, SP1, XAB2, CDC2L1, MSX1, and AQP3) plus Na/K ATPase, a gene previously known to be involved in blastocoe formation, was studied by quantitative real-time PCR (QPCR) in 6 replicate pools of 5 embryos produced by maturation, fertilization, and embryo culture in vitro. Stages studies included immature and mature oocyte, zygote, 2- cell, 4-cell, 8-cell, 16-cell, morula, blastocyst, and hatched blastocyst. In addition, in vivo derived Day 13 and Day 16 embryos were included as controls to confirm down-regulation after Day 7. Data were analysed using the GLM procedure of SAS. The QPCR expression data supported the RNA Seq data in that expression of all transcripts was downregulated after the blastocyst stage. Expression before the blastocyst stage was characterised by 1 of 3 broad patterns: (1) the expression was of maternal origin where the expression was very high up to 8-cell stage and decreased subsequently (MSX1), (2) the expression was of embryonic origin being low up to the 8-cell stage and increasing thereafter (TAF8, ESRRB, AQP3, and Na/K ATPase), or (3) static or decreased expression from oocyte to the maternal-zygotic transition followed by increased expression from the 16-cell stage (GINS1, NCAPG2, SP1, XAB2, and CDC2L1). In conclusion, the genes identified in this cluster, despite having different patterns of expression before the blastocyst stage, may represent markers of blastocyst formation in cattle given their downregulation subsequently. Supported by Science Foundation Ireland (07/SRC/B1156).

Identification of Early Zygotic Genes in the Yellow Fever Mosquito Aedes aegypti and Discovery of a Motif Involved in Early Zygotic Genome Activation

During early embryogenesis the zygotic genome is transcriptionally silent and all mRNAs present are of maternal origin. The maternal-zygotic transition marks the time over which embryogenesis changes its dependence from maternal RNAs to zygotically transcribed RNAs. Here we present the first systematic investigation of early zygotic genes (EZGs) in a mosquito species and focus on genes involved in the onset of transcription during 2–4 hr. We used transcriptome sequencing to identify the “pure” (without maternal expression) EZGs by analyzing transcripts from four embryonic time ranges of 0–2, 2–4, 4–8, and 8–12 hr, which includes the time of cellular blastoderm formation and up to the start of gastrulation. Blast of 16,789 annotated transcripts vs. the transcriptome reads revealed evidence for 63 (P<0.001) and 143 (P<0.05) nonmaternally derived transcripts having a significant increase in expression at 2–4 hr. One third of the 63 EZG transcripts do not have predicted introns compared to 10% of all Ae. aegypti genes. We have confirmed by RT-PCR that zygotic transcription starts as early as 2–3 hours. A degenerate motif VBRGGTA was found to be overrepresented in the upstream sequences of the identified EZGs using a motif identification software called SCOPE. We find evidence for homology between this motif and the TAGteam motif found in Drosophila that has been implicated in EZG activation. A 38 bp sequence in the proximal upstream sequence of a kinesin light chain EZG (KLC2.1) contains two copies of the mosquito motif. This sequence was shown to support EZG transcription by luciferase reporter assays performed on injected early embryos, and confers early zygotic activity to a heterologous promoter from a divergent mosquito species. The results of these studies are consistent with the model of early zygotic genome activation via transcriptional activators, similar to what has been found recently in Drosophila.

Transcriptome-wide analysis of small RNA expression in early zebrafish development

During early vertebrate development, a large number of noncoding RNAs are maternally inherited or expressed upon activation of zygotic transcription. The exact identity, expression levels, and function for most of these noncoding RNAs remain largely unknown. miRNAs (microRNAs) and piRNAs (piwi-interacting RNAs) are two classes of small noncoding RNAs that play important roles in gene regulation during early embryonic development. Here, we utilized next-generation sequencing technology to determine temporal expression patterns for both miRNAs and piRNAs during four distinct stages of early vertebrate development using zebrafish as a model system. For miRNAs, the expression patterns for 198 known miRNAs within 122 different miRNA families and eight novel miRNAs were determined. Significant sequence variation was observed at the 5' and 3'ends of miRNAs, with most extra nucleotides added at the 3' end in a nontemplate directed manner. For the miR-430 family, the addition of adenosine and uracil residues is developmentally regulated and may play a role in miRNA stability during the maternal zygotic transition. Similar modification at the 3' ends of a large number of miRNAs suggests widespread regulation of stability during early development. Beside miRNAs, we also identified a large and unexpectedly diverse set of piRNAs expressed during early development.

The RNA-binding protein XSeb4R regulates maternal Sox3 at the posttranscriptional level during maternal-zygotic transition in Xenopus

The maternal-zygotic transition (MZT) is an embryonic event that overlaps with and plays key roles in primary germ layer specification in vertebrates. During MZT, maternally supplied mRNAs are degraded while zygotic transcripts are synthesized to either reinforce the already specified cell fate or to trigger new cell identity. Here, we show that forced expression of the RNA-binding protein, XSeb4R, in animal pole blastomeres of Xenopus embryos, inappropriately stabilizes transcripts there, including maternal Sox3. This leads to the impaired ability of the ectodermal progenitors to respond to factors regulating brain patterning and their eventual loss by apoptosis. XSeb4R protein binds specifically to the 3′UTR of Sox3 mRNA. XSeb4R gain-of-function in ectodermal explants reveals increased stability of the maternal Sox3 transcripts, associated with a robust Sox3 protein production. Conversely, whereas XSeb4R depletion abolishes VegT expression, the amount of the maternal Sox3 mRNA is rather increased but without augmentation in the amount of Sox3 protein. Moreover, XSeb4R protein knockdown leads to the modification of the ectoderm–mesoderm boundary, marked by expanded/shifted expression of the mesodermal marker genes such as Xbra and Apod, followed by an expression inhibition of Epi. K., an ectodermal marker. Overall, our data suggest XSeb4R as a novel player in gene expression regulation, acting at the posttranscriptional level during ectoderm specification in Xenopus.

158 RELATIONSHIP BETWEEN FOURTH CELL CYCLE DURATION AND POST-TRANSFER VIABILITY IN IN VITRO-FERTILIZED BOVINE EMBRYOS

In cattle, the prediction of embryonic viability after embryo transfer is an important research target. A previous study has indicated that the duration of the fourth cell cycle at the time of maternal-zygotic transition, which is involved in in vitro embryonic development, may be an indicator of blastocyst formation; this study showed that embryos with a short fourth cell cycle have a better potential of developing into blastocysts than those with a long fourth cell cycle (Lequarre et al. 2003 Biol. Reprod. 69, 1707–1713). However, the relationship between the fourth cell cycle duration and post-transfer viability of embryos is unclear. The aim of the present study was to examine the effect of the fourth cell cycle duration on embryo development after embryo transfer. Twenty-five IVF bovine embryos were cultured in well-of-the-well culture dishes contained 125 μ of CR1aa supplemented with 5% calf serum at 38.5°C in 5% O2 and 5% CO2 for 168 h after insemination. In vitro development of the embryos was monitored using time-lapse cinematography (Sugimura et al. 2010 Biol. Reprod. 83, 970–978). We found that 61% of the blastocysts had a long fourth cell cycle (41.5 ± 5.9 h), which is commonly referred to as the lag phase, whereas the remaining embryos had a short fourth cell cycle (7.4 ± 4.5 h). All the embryos with a short fourth cell cycle exhibited a lag phase in the next cell cycle (32.9 ± 6.6 h). Moreover, embryos with a short fourth cell cycle were found to have a higher blastocyst rate (75.8%) than those with a long fourth cell cycle (48.1%; Student's t-test, P &lt; 0.01). However, embryonic cell number, apoptosis incidence, chromosomal abnormality and O2 consumption were found to be identical between the 2 groups (Student's t-test, P &gt; 0.05). Real-time reverse-transcription PCR results of the individual blastocysts showed that the relative expression of 5 genes related to pregnancy reorganization, placentation and fetal growth—namely, CDX2, IFN-τ, PLAC8, AKR1B1 and IGF2R—did not differ between the 2 groups (Student's t-test, P &gt; 0.05). Furthermore, blastocysts derived from embryos with long (n = 30) and short (n = 19) fourth cell cycles were transferred into 49 recipient cows; we did not observe any difference between the long and short fourth cell cycles on the rates of pregnancy (long vs short fourth cell cycle, 30.0 vs 52.6%) and delivery (long vs short fourth cell cycle, 30.0 vs 47.4%; Yates' corrected chi-square test, P &gt; 0.10). These results show that blastocysts derived from embryos with either long or short fourth cell cycles have identical developmental competence after embryo transfer. Therefore, the fourth cell cycle duration during maternal-zygotic transition appears to be unavailable as the indicator of post-transfer viability of IVF bovine embryos. This work was supported by the Research and Developmental Program for New Bio-Industry Initiatives.

Clusters of Temporal Discordances Reveal Distinct Embryonic Patterning Mechanisms in Drosophila and Anopheles

Author SummaryLinking genotype to phenotype is a major undertaking in modern biological research. A variety of strategies are used but have generally failed to explain the maintenance and acquisition of new phenotypic traits in changing populations. We propose that whole-genome cross-species comparisons can be used to identify gene clusters underlying phenotypic variation. In the present study we used gene expression datasets collected during fruit fly and mosquito embryogenesis to identify temporal changes in gene expression. We found that differentially represented tissue types (such as extraembryonic serosa) were clearly manifested by clusters of local discordances in gene expression. Discordances were also observed for a suite of maternally expressed genes, consistent with the notion that the abrupt maternal-zygotic transition seen in Drosophila is an evolutionary innovation of higher Diptera. We propose that gene clustering by expression discordance can be used to determine the genetic basis of phenotypic variation.

Non-coding RNAs as regulators of embryogenesis

Non-coding RNAs (ncRNAs) are emerging as key regulators of embryogenesis. They control embryonic gene expression by several means, ranging from microRNA-induced degradation of mRNAs to long ncRNA-mediated modification of chromatin. Many aspects of embryogenesis seem to be controlled by ncRNAs, including the maternal-zygotic transition, the maintenance of pluripotency, the patterning of the body axes, the specification and differentiation of cell types and the morphogenesis of organs. Drawing from several animal model systems, we describe two emerging themes for ncRNA function: promoting developmental transitions and maintaining developmental states. These examples also highlight the roles of ncRNAs in ensuring a robust commitment to one of two possible cell fates.

Granzyme Gis expressed in the two-cell stage mouse embryo and is required for the maternal-zygotic transition

BackgroundDetailed knowledge of the molecular and cellular mechanisms that direct spatial and temporal gene expression in pre-implantation embryos is critical for understanding the control of the maternal-zygotic transition and cell differentiation in early embryonic development. In this study, twenty-three clones, expressed at different stages of early mouse development, were identified using differential display reverse transcription polymerase chain reaction (DDRT-PCR). One of these clones, which is expressed in 2-cell stage embryos at 48 hr post-hCG injection, shows a perfect sequence homology to the gene encoding the granzyme G protein. The granzyme family members are serine proteases that are present in the secretory granules of cytolytic T lymphocytes. However, the pattern of granzyme G expression and its function in early mouse embryos are entirely unknown.ResultsUpon the introduction of an antisense morpholino (2 mM) against granzyme G to knock-down endogenous gene function, all embryos were arrested at the 2- to 4-cell stages of egg cleavage, and the de novo synthesis of zygotic RNAs was decreased. The embryonic survival rate was dramatically decreased at the late 2-cell stage when serine protease-specific inhibitors, 0.1 mM 3,4-dichloroisocoumarin (3,4-DCI), and 2 mM phenyl methanesulphonyl fluoride (PMSF), were added to the in vitro embryonic culture medium. Survival was not affected by the addition of 0.5 mM EDTA, a metalloproteinase inhibitor.ConclusionWe characterized for the first time the expression and function of granzyme G during early stage embryogenesis. Our data suggest that granzyme G is an important factor in early mouse embryonic development and may play a novel role in the elimination of maternal proteins and the triggering of zygotic gene expression during the maternal-zygotic transition.

Chromatin signature of embryonic pluripotency is established during genome activation

After fertilization the embryonic genome is inactive until transcription is initiated during the maternal-zygotic transition. This transition coincides with the formation of pluripotent cells, which in mammals can be used to generate embryonic stem cells. To study the changes in chromatin structure that accompany pluripotency and genome activation, we mapped the genomic locations of histone H3 molecules bearing lysine trimethylation modifications before and after the maternal-zygotic transition in zebrafish. Histone H3 lysine 27 trimethylation (H3K27me3), which is repressive, and H3K4me3, which is activating, were not detected before the transition. After genome activation, more than 80% of genes were marked by H3K4me3, including many inactive developmental regulatory genes that were also marked by H3K27me3. Sequential chromatin immunoprecipitation demonstrated that the same promoter regions had both trimethylation marks. Such bivalent chromatin domains also exist in embryonic stem cells and are thought to poise genes for activation while keeping them repressed. Furthermore, we found many inactive genes that were uniquely marked by H3K4me3. Despite this activating modification, these monovalent genes were neither expressed nor stably bound by RNA polymerase II. Inspection of published data sets revealed similar monovalent domains in embryonic stem cells. Moreover, H3K4me3 marks could form in the absence of both sequence-specific transcriptional activators and stable association of RNA polymerase II, as indicated by the analysis of an inducible transgene. These results indicate that bivalent and monovalent domains might poise embryonic genes for activation and that the chromatin profile associated with pluripotency is established during the maternal-zygotic transition.

Evolutionary analysis of the kinesin light chain genes in the yellow fever mosquito Aedes aegypti: gene duplication as a source for novel early zygotic genes

BackgroundThe maternal zygotic transition marks the time at which transcription from the zygotic genome is initiated and a subset of maternal RNAs are progressively degraded in the developing embryo. A number of early zygotic genes have been identified in Drosophila melanogaster and comparisons to sequenced mosquito genomes suggest that some of these early zygotic genes such as bottleneck are fast-evolving or subject to turnover in dipteran insects. One objective of this study is to identify early zygotic genes from the yellow fever mosquito Aedes aegypti to study their evolution. We are also interested in obtaining early zygotic promoters that will direct transgene expression in the early embryo as part of a Medea gene drive system.ResultsTwo novel early zygotic kinesin light chain genes we call AaKLC2.1 and AaKLC2.2 were identified by transcriptome sequencing of Aedes aegypti embryos at various time points. These two genes have 98% nucleotide and amino acid identity in their coding regions and show transcription confined to the early zygotic stage according to gene-specific RT-PCR analysis. These AaKLC2 genes have a paralogous gene (AaKLC1) in Ae. aegypti. Phylogenetic inference shows that an ortholog to the AaKLC2 genes is only found in the sequenced genome of Culex quinquefasciatus. In contrast, AaKLC1 gene orthologs are found in all three sequenced mosquito species including Anopheles gambiae. There is only one KLC gene in D. melanogaster and other sequenced holometabolous insects that appears to be similar to AaKLC1. Unlike AaKLC2, AaKLC1 is expressed in all life stages and tissues tested, which is consistent with the expression pattern of the An. gambiae and D. melanogaster KLC genes. Phylogenetic inference also suggests that AaKLC2 genes and their likely C. quinquefasciatus ortholog are fast-evolving genes relative to the highly conserved AaKLC1-like paralogs. Embryonic injection of a luciferase reporter under the control of a 1 kb fragment upstream of the AaKLC2.1 start codon shows promoter activity at least as early as 3 hours in the developing Ae. aegypti embryo. The AaKLC2.1 promoter activity reached ~1600 fold over the negative control at 5 hr after egg deposition.ConclusionsTranscriptome profiling by use of high throughput sequencing technologies has proven to be a valuable method for the identification and discovery of early and transient zygotic genes. The evolutionary investigation of the KLC gene family reveals that duplication is a source for the evolution of new genes that play a role in the dynamic process of early embryonic development. AaKLC2.1 may provide a promoter for early zygotic-specific transgene expression, which is a key component of the Medea gene drive system.

Before Maternal-Zygotic Transition … There Was Morphogenetic Function of Nuclei

The idea of a major developmental transition that includes the activation of the embryonic genome has a long history. In the 1950-1960s this concept was developed to a large extent due to the efforts of Alexander Neyfakh, who described a specific type of deleterious effect resulting from X-ray irradiation of fish eggs. He interpreted the radiation-sensitive target as the nucleus and established the onset of the function of the zygotic genome, naming it the morphogenetic function of nuclei, what we call now the midblastula transition. Most of his studies were performed using the loach (Misgurnus fossilis), a European teleost. Neyfakh's efforts paved the way to understanding the whole phenomenon of the maternal-zygotic transition.

Maternal Argonaute 2 Is Essential for Early Mouse Development at the Maternal-Zygotic Transition

Activation of zygotic gene expression in the two-cell mouse embryo is associated with destruction of maternally inherited transcripts, an important process for embryogenesis about which little is understood. We asked whether the Argonaute (Ago)/RNA-induced silencing complex, providing the mRNA "slicer" activity in gene silencing, might contribute to this process. Here we show that Ago2, 3, and 4 transcripts are contributed to the embryo maternally. By systematic knockdown of maternal Ago2, 3, and 4, individually and in combination, we find that only Ago2 is required for development beyond the two-cell stage. Knockdown of Ago2 stabilizes one set of maternal mRNAs and reduces zygotic transcripts of another set of genes. Ago2 is localized in mRNA-degradation P-bodies analogous to those that function in RNAi-like mechanisms in other systems. Profiling the expression of microRNAs throughout preimplantation development identified several candidates that could potentially work with Ago2 to mediate degradation of specific mRNAs. However, their low abundance raises the possibility that other endogenous siRNAs may also participate. Together, our results demonstrate that maternal expression of Ago2 is essential for the earliest stages of mouse embryogenesis and are compatible with the notion that degradation of a proportion of maternal messages involves the RNAi-machinery.

Erasure of the paternal transcription program during spermiogenesis: The first step in the reprogramming of sperm chromatin for zygotic development

Male germ cells possess a unique epigenetic program and express a male-specific transcription profile. However, when its chromatin is passed onto the zygote, it expresses an transcription/epigenetic program characteristic of the zygote. The mechanism underlying this reprogramming process is not understood at present. In this study, we show that an extensive range of chromatin factors (CFs), including essential transcription factors and regulators, remodeling factors, histone deacetylases, heterochromatin-binding proteins, and topoisomerases, were removed from chromatin during spermiogenesis. This process will erase the paternal epigenetic program to generate a relatively naive chromatin, which is likely to be essential for installation of the zygotic developmental program after fertilization. We have also showed that transcription termination in male germ cells was temporally correlated with CF dissociation. A genome-wide CF dissociation will inevitably disassemble the transcription apparatus and regulatory mechanism and lead to transcription silence. Based on data presented in this and previous studies (Sun et al., Cell Research [2007] 17:117-134), we propose that paternal-zygotic transcription reprogramming begins with a genome-wide CF dissociation to erase the existing transcription program in later stages of spermatogenesis. This will be followed by assembling of the zygotic equivalent after fertilization. The transcription/epigenetic program of the male germ cell is transformed into a zygotic one using an erase-and-rebuild strategy similar to that used in the maternal-zygotic transition. It is also noted that transcription is terminated long after meiosis is completed and before chromatin becomes highly condensed during spermatogenesis. The temporal order of these events suggests that transcription silence does not have to be coupled to meiosis or chromatin condensation.

The Maternal-Zygotic Transition: Death and Birth of RNAs

Maternal gene products drive early development when the newly formed embryo is transcriptionally inactive. During the maternal-zygotic transition, embryonic transcription is initiated and many maternal RNAs are degraded. Multiple mechanisms regulate the birth of zygotic RNAs and the death of maternal RNAs. Genome activation appears to rely in part on the sequestration of transcriptional repressors by the exponentially increasing amount of DNA during cleavage divisions. Maternal RNA degradation is induced by the binding of proteins and microRNAs to the 3' untranslated region of target RNAs.

The Transitioning Germ Line

![Figure][1] CREDIT: GETTY IMAGES/VISUALS UNLIMITED Germline constancy is a must for species continuity, but the life of the germ cell itself is full of change. The trek to and from the germ line includes numerous transitions and decisions deftly choreographed for the gametes' unique ability in nature to develop into a complex animal and pass on genetic information. This special section highlights the transitioning germ line and otherwise expands on intriguing aspects of these cells of heredity. In the early embryo, cells decide between becoming soma or germ line. Mechanisms protect germ cells from a somatic fate, just as somatic cells require insulation from the germline differentiation pathway. Strome and Lehmann (p. [392][2]) outline activities in the fly and worm, such as transcriptional quiescence and inheritance of germ cell determinants, and Hayashi et al. (p. [394][3]) tell of the murine system, invoking an inductive signal. Although model systems use different strategies, common themes exist. A new class of molecules reportedly participates in germ cell development. Lin (p. [397][4]) describes the generation and role of germline-specific small RNAs termed piRNAs. Functions as diverse as posttranscriptional regulation and epigenetic programming are suggested. Another epigenetic event—de novo DNA methylation—silences imprinted genes and repeat elements during vertebrate gametogenesis. Schaefer et al. (p. [398][5]) detail this heritable silencing mechanism and its sexual dimorphism. As the germ cell transitions to the mature form, more decisions are made: to enter mitosis or meiosis and to differentiate as sperm or egg, with possible linkage of these decisions as proposed by Kimble and Page (p. 400). Stem cell self-renewal versus differentiation in the Drosophila male and female germ lines is discussed by Fuller and Spradling (p. [402][6]). Switching to vertebrate germline stem cells, Brinster (p. [404][7]) reports methods for the recovery, culture, and transplantation of spermatogonial stem cells; and Daley (p. 409) summarizes advances in embryonic stem cell (ESC) differentiation to a germline fate: procedures with research and clinical applications but not without limitations. In an Editorial, McLaren (p. [339][8]) takes the discussion further, highlighting ethical and social issues surrounding ESC-derived germ cells. Changes continue as the oocyte transitions to the fertilized egg. Stitzel and Seydoux (p. 407) detail the oocyte-to-zygote transition, with associated alterations in protein synthesis, protein and RNA degradation, and organelle remodeling; and Schier (p. 406) discusses zygotic genome silencing and maternal messenger RNA degradation around the maternal-zygotic transition. In News, Travis (p. 390) discusses “urbisexuality” with a biologist pondering the evolution of germ cell specification, and Leslie (p. 388) details how oocyte freezing has become more effective and acceptable despite questions about the long-term health of offspring. Finally, in [ Science 's STKE][9], McClure discusses how poppies reject self-pollen, and Boldajipour and Raz highlight similarities in Drosophila and mouse mechanisms involved in the migration of primordial germ cells and elimination of ectopic cells. The articles here give but a sampling of exciting ongoing research on the seeds of life—themselves products of change. Advances bear fruit in understanding development and its underlying genetics, as well as infertility. [1]: pending:yes [2]: /lookup/doi/10.1126/science.1140846 [3]: /lookup/doi/10.1126/science.1137545 [4]: /lookup/doi/10.1126/science.1137543 [5]: /lookup/doi/10.1126/science.1137544 [6]: /lookup/doi/10.1126/science.1140861 [7]: /lookup/doi/10.1126/science.1137741 [8]: /lookup/doi/10.1126/science.1142267 [9]: http://www.sciencemag.org/sciext/germcells/#section_in-stke

The effect of glucosamine concentration on the development and sex ratio of bovine embryos

Glucosamine is a component of hyaluronic acid and an alternative substrate to glucose for the extracellular matrix synthesis of COCs. Its addition to an IVM medium reduces the glucose consumption of bovine COCs. Glucosamine is also metabolized to UDP- N-acetyl glucosamine (UDP-GlcNAc) via the hexosamine biosynthesis pathway and is utilized for O-linked glycosylation by the X-linked enzyme, O-linked GlcNAc transferase (OGT). Moreover, the inactivation of the second X chromosome in female embryos is influential in producing the sex ratio bias observed in vitro when embryos are cultured in the presence of glucose above 2.5 mM. Accordingly, the aim of this study is to examine whether the presence of glucosamine during maturation or embryo culture causes a sex ratio bias in bovine blastocysts. Glucosamine was added to the medium in three different embryo developmental periods: in vitro maturation, the one-cell to eight-cell stage (before the maternal–zygotic transition, MZT), and the eight-cell to blastocyst stage (after MZT). When glucosamine was added during in vitro maturation, the developmental competence of oocytes was severely compromised. However, the sex ratio of embryos was not influenced. When glucosamine was added to embryo culture medium during development from one-cell to eight-cell stage (before MZT), it affected neither the development nor the sex ratio of bovine embryos. Finally, when glucosamine was added after MZT, the development rate of embryos was severely decreased, and the sex ratio was skewed toward males. Moreover, an inhibitor of OGT, benzyl-2-acetamido-2-deoxy-α- d-galactopyranoside (BADGP), negated the effect of glucosamine on the sex ratio when it was added to embryo culture medium from the eight-cell to blastocyst stage (after MZT). These results suggest that, like glucose, the supplementation of glucosamine into the medium skewed the sex ratio to males and that OGT, an X-linked enzyme, was involved in this phenomenon. Moreover, this effect of glucosamine was limited only to when it was present in the embryo culture medium after MZT.

The influence of follicle size, FSH‐enriched maturation medium, and early cleavage on bovine oocyte maternal mRNA levels

Transcription is arrested in the bovine oocyte within the first few hours of in vitro maturation, thus the stored maternal mRNAs accumulated in the oocyte are essential to sustain development until the Maternal-Zygotic Transition. In vivo matured oocytes have superior blastocyst formation rates than in vitro matured oocytes, suggesting that the mRNA content of these oocytes is of higher quality. To determine which transcripts may be associated with developmental competence, a Suppressive Subtractive Hybridization was performed between oocytes collected by ovariectomy at 6 hr post-LH surge and oocytes from slaughterhouse collected after 6 hr of maturation, resulting in a library enriched in these functionally important mRNAs. The clones were spotted onto a cDNA microarray and transcripts potentially associated with developmental competence were hybridized onto these slides. Hybridizations were performed with transcripts up-regulated in oocytes cultured for 6 hr in the presence or absence of rFSH in vitro, and secondly with transcripts up regulated in early-cleaving embryos versus those at the one-cell stage at 36 hr postfertilization. From these hybridizations, 13 candidates were selected. Their functional association with embryonic competence was validated by measuring their relative transcript levels by quantitative real-time PCR in eight different conditions: oocytes cultured with or without rFSH, early--versus late-cleaving embryos, and oocytes from different follicle sizes (1-3, 3-5, 5-8, and >8 mm of diameter). The gene candidates CCNB2, PTTG1, H2A, CKS1, PSMB2, SKIIP, CDC5L, RGS16, and PRDX1 showed a significant quantitative association with competence compared to BMP15, GDF9, CCNB1, and STK6.

Cell death in development: shaping the embryo

Cell death in animals is normally classified as type I (apoptotic), type II (autophagic) or necrotic. Of the biologically controlled types of death, in most embryos apoptosis is the most common, although in metamorphosis and in cells with massive cytoplasm type II is often seen, and intermediate forms are seen. For vertebrate embryos other than mammals, apoptosis is not seen prior to gastrulation but thereafter is used to sculpt the organs of the embryo, while overproduction of cells with subsequent death of excess cells is a common means of generating high specificity with low information cost. In zebrafish at least, the inability of embryos prior to the maternal-zygotic transition to undergo apoptosis appears to derive from the inability of the cells to resist lysis once apoptosis begins, rather than any inhibition of apoptosis. In mammalian embryos, apoptosis is seen during cavitation. Thereafter, as in other embryos, cell death plays a major role in shaping and sculpting the embryo. In those situations that have been carefully studied, cell death is under tight genetic control (including regulation of gene products whose function in cell death is not yet known, such as cdk5), with activation of apoptosis sometimes regulated by local environmental variables.

148 EFFECT OF FOLLISTATIN TREATMENT POST-FERTILIZATION ON TIME TO FIRST CLEAVAGE, DEVELOPMENT TO THE BLASTOCYST STAGE, AND CELL ALLOCATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

Previous studies from our laboratory have demonstrated a positive association of follistatin mRNA abundance with oocyte competence. Follistatin mRNA is greater in germinal vesicle stage oocytes collected from prepubertal (model of poor oocyte competence) vs. adult animals. Furthermore, follistatin mRNA abundance is also greater in early-cleaving 2-cell bovine embryos (collected prior to the maternal zygotic transition and initiation of significant transcription from the embryonic genome) than their late-cleaving counterparts. Given these results and the fact that early-cleaving embryos develop to the blastocyst stage at a greater rate, we hypothesized that follistatin has a stimulatory role in early embryonic development. To begin to test this hypothesis, we determined the effects of follistatin treatment of in vitro-produced bovine embryos (during the initial 72 h post-fertilization) on time to first cleavage, development to the blastocyst stage (Day 7), and blastocyst cell allocation (quality). Cumulus–oocyte complexes (COCs) were harvested from ovaries obtained from a local abattoir, matured, and fertilized in vitro. After 20 h of co-incubation with spermatozoa, presumptive zygotes were stripped of cumulus cells and cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10, or 100 ng mL-1 follistatin (n = 25 presumptive zygotes per treatment; n = 6 replicates). Proportions of embryos reaching the 2-cell stage within 30 h (early-cleaving), 30–36 h (late-cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Embryos at the 8–16-cell stage were separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until Day 7. The proportion of embryos reaching the blastocyst stage at Day 7 post-fertilization was recorded and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells determined by differential staining. Follistatin treatment did not increase the rate of total cleavage and the proportion of late-cleaving embryos when compared to control. However, supplementation with 1 and 10, but not 100, ng mL-1 follistatin increased the proportion of early-cleaving embryos (26.3 and 35.3% vs. 9.5%) and development to the blastocyst stage (28.6 and 31.7% vs. 18.4%) relative to controls (P &amp;lt; 0.05). Treatment with 10 ng mL-1 follistatin increased total cell numbers (130.1 vs. 110.9) and proportion of trophectoderm cells (61.6% vs. 48.4%) and decreased the ICM/total cell ratio (38.4% vs. 51.5%) in Day 7 blastocysts relative to controls (P &amp;lt; 0.05). The results indicate that exogenous follistatin treatment during the early stages of in vitro bovine embryo development can enhance time to first cleavage, development to the blastocyst stage, and cell allocation in favor of increased trophectoderm cells, and can support a potential functional role for follistatin in early embryogenesis.

Transcription factor expression patterns in bovine in vitro-derived embryos prior to maternal-zygotic transition.

Maternal-zygotic transition (MZT) is a complex phenomenon characterized by the initiation of transcription in the embryo and the replacement of maternal mRNA with embryonic mRNA. In order for this to occur, transcriptional activation requires various factors and conditions. Our hypothesis is that the lack of transcription in the bovine pre-MZT embryo is due, in part, to an incomplete or dormant transcriptional apparatus. Therefore, in accordance with this hypothesis, functioning transcriptional mechanisms should appear in the eight-cell bovine embryo to facilitate embryonic transcription during the MZT. With this in mind, we examined the presence of selected transcription factors during preimplantation embryo development to establish how their transcript levels change in bovine pre-MZT embryos. To achieve this goal, real-time reverse transcription-polymerase chain reaction was used to quantify the mRNA level of several different transcription factors (YY1, HMGA1, RY-1, P300, CREB, YAP65, HMGN1, HMGB1, NFAR, OCT-4, TEAD2, ATF-1, HMGN2, MSY2, and TBP) in germinal vesicle (GV) and metaphase II (MII) bovine oocytes and in two-, four-, eight-cell, and blastocyst stage embryos produced in vitro. Our results demonstrate that all genes examined can be grouped into five different categories according to their mRNA expression patterns at the developmental stages observed. To summarize, all transcription factors studied were present in pre-MZT embryos and the expression pattern of many of them suggest a potential role in MZT.